Kaposi's sarcoma (KS) is the most common neoplasm in patients with acquired immunodeficiency syndrome (AIDS). Human herpesvirus 8, or Kaposi's sarcoma-associated herpesvirus (KSHV), has been implicated in all forms of KS, as well as in primary effusion lymphoma and some forms of Castleman's disease. Central to pathogenesis and lytic KSHV infection is the viral replication, including the well-coordinated assembly process leading to subviral particles (pro-, A-, B-, C-capsids) and infectious virions. Using electron cryomicroscopy (cryoEM) techniques well established during his previous structural studies of other herpesviruses, the applicant has published the first three-dimensional (3D) structure of KSHV B-capsids purified from BCBL-1 cells. This structure, while revealing a capsid shell almost identical to those of other herpesviruses, nevertheless exhibited striking differences at the outermost region of the capsid. This observation suggests that KSHV and other herpesviruses share a similar mechanism of capsid assembly, but virion proteins situated at the outer radial regions, such as pORF65 and the tegument proteins, may have KSHV specific assembly/functional roles. The proposed studies are directed at testing this hypothesis by determining and understanding the molecular interactions essential to KSHV capsid and tegument assembly. The goals of this application are (1) to determine the structural basis of KSHV assembly and molecular interactions by determining a high-resolution (-7 A) structure of the KSHV B-capsid using cryoEM reconstruction techniques well established by the applicant's group; (2) to locate pORF65 and other capsid proteins and visualize their antibody epitopes; (3) to elucidate the mechanisms whereby capsid proteins interact with both tegument proteins and viral DNA by determining the structure of the intact KSHV virion and comparing it with other herpesviruses; (4) to determine the structural basis for KSHV viral assembly and morphogenesis by structural comparisons of KSHV B-capsid and protease-minus capsids (procapsids), which are generated by ribozyme-mediated, KSHV protease-specific inhibition. The results of these experiments will lead to a better understanding of the process of KSHV lytic replication and the identification of potential targets for therapeutic intervention.